There are many protein purification method has been disclosed, such as shown in U.S. Pat. No. 5,278,284, issued in 1994 to Lusk et al, entitled “Protein Purification Method”, U.S. Pat. No. 6,036,861, issued in 2000 to Flickinger et al, entitled “Protein adsorption by very dense porous zirconium oxide particles in expanded beds”, U.S. Pat. No. 5,837,826, issued in 1998 to Flickinger et al, entitled “Protein adsorption by very dense porous zirconium oxide particles in expanded beds”, U.S. Pat. No. 7,026,453, issued in 2006 to Haj-Ahmad, entitled “Method of protein purification”, and Taiwan Patent No. 365,540, issued in 1999, to Adames Omar et al, entitled “A method for the removal of viruses from protein solutions by adsorption using solid phase suspension particles”. Taiwan Patent No. 365,540 disclosed that adsorbent is selected from the group silicone, Zirconium oxide particles, diatomaceous and Silicon carbon compounds, and subsequently the solid phase is separated from the liquid phase by extraction to obtain the protein purification.
In addition, as shown in U.S. Pat. No. 6,339,142, issued in 2002 to Basey et al, entitled “Protein Purification”, or in Taiwan Patent No. 128021, issued in 1990 to David Naveh, entitled “Method of Purifying Protein”, or in Taiwan Publication No 201117883, published on 2011 to Chem, Ming Kai, entitled “Method For Protein Purification”. Taiwan Publication No. 201117883 disclosed that the protein purification is performed by using an ion exchange resin and adjusting pH value of the buffer solution according to the different isoelectric point (PI) protein aqueous to achieve the effect of the protein purification.
In addition, as shown in Taiwan Patent No. 169,356, issued in 1991 to Staples et al, entitled “Process For Purifying a Protein” which disclosed a process for the purification of proteins from solutions containing contaminants of similar net charge and molecular weight is provided, comprising contacting a solution containing the desired protein with an immobilized metal affinity chromatography resin in a buffer containing a low concentration of a weak ligand for the chelant of the resin. In addition, as shown in Taiwan Publication No. 200300173, published on 2003 to Minoshima, entitled “Process For Producing Concentrated/Purified Protein Using Clay Mineral Composition” which disclosed a method for isolating a protein characterized in that a protein adsorbed on a clay mineral-containing composition is isolated using at least one member selected from the group consisting of fatty acid esters whose fatty acid residue has a carbon atom number of not more than 30. In Minoshima, utilized the ion exchange, molecular sieving effect, gel filtration chromatography or gel filtration chromatography with ampholine to purify the protein. Generally, the typical analysis method is isoelectric focusing (IEF). For separating, concentrating and purifying the crude protein aqueous, the crude protein aqueous is subjected to the desalination treatment and concentration treatment. Thus, the purification of the protein aqueous is quite complex and requires highly cost for the industrial scale, and the technical problems is also involved.
Minoshima also disclosed that a protein is adsorbed on a clay mineral and there have been known techniques for separating the protein from the clay mineral in which the protein adsorbed on the clay mineral is isolated through the use of an eluent having a different pH value or an ionic strength, but these techniques are accompanied with an abrupt change of pH or ionic strength, which may cause various changes in the higher-order structure of the protein strictly involved in the functionality thereof.
In addition, Chern, Ming Kai disclosed that ion exchange chromatography can apply for protein purification. In present technology, the ion exchange chromatography utilizes a sample with the protein which is loaded into a column with ion exchanger, and the charged groups on the protein and immobilized group of the ion exchange agent are interacted, such that the protein is absorbed on the ion exchange agent in the column, the above method can be called equilibrium model.
Next, the salt solution with increasing gradient concentration is imported in the same direction as sample with the protein. Thus, the protein with the degree of interaction which is different from the ion exchange agent that will be eluted sequentially in different salt concentrations, in which based on the prior art of ion exchange chromatography lacks of specific affinity. Thus, when ion exchange chromatography is used to purify a specific target protein, the target protein is to be separated from the wash solution by combined with other purification process, such as affinity chromatography. Thus, the purification process requires genetic engineering or the preparation of specific antibodies, and the cost of the purification would be increased.
Furthermore, in the field of analytical chemistry, for the separation of the inorganic species which develops a kind of reverse elution method, where it is inverted by simple elution flow direction of the sample loading to reduce the peak dispersion effect, and the problem of the irreversibility of the sample ions ion exchange (irreversible ion exchange) can be avoided, and the dip peak can also be reduced. Specifically, the sample is imported in the opposite direction to the direction of the elution, and is compressed to form a compact band by the initial stage of elution of the sample ions, such that the band broadening of the concentrator column will be minimized. Because the treating direction will influence the effect of separation, it is not only a balanced the interaction, and non-balanced interaction can also play an important role in the aforementioned method.
However, for comparing with the inorganic ions, the protein molecules are consisted by a plurality of amino acid molecules. According to the currently protein purification, the problems of the peak dispersion still exist. The fraction is separated from the salt concentration range of elution gradient which is further purified to obtain more protein with homogeneity. Therefore, the conventional prior art still lacks of disclosing a method for separating the protein with high homogeneity.